Inhalation of ACE2-expressing lung exosomes provides prophylactic protection against SARS-CoV-2

Continued emergence of SARS-CoV-2 variants of concern that are capable of escaping vaccine-induced immunity highlights the urgency of developing new COVID-19 therapeutics. An essential mechanism for SARS-CoV-2 infection begins with the viral spike protein binding to the human ACE2. Consequently, inhibiting this interaction becomes a highly promising therapeutic strategy against COVID-19. Herein, we demonstrate that ACE2-expressing human lung spheroid cells (LSC)-derived exosomes (LSC-Exo) could function as a prophylactic agent to bind and neutralize SARS-CoV-2, protecting the host against SARS-CoV-2 infection. Inhalation of LSC-Exo facilitates its deposition and biodistribution throughout the whole lung in a female mouse model. We show that LSC-Exo blocks the interaction of SARS-CoV-2 with host cells in vitro and in vivo by neutralizing the virus. LSC-Exo treatment protects hamsters from SARS-CoV-2-induced disease and reduced viral loads. Furthermore, LSC-Exo intercepts the entry of multiple SARS-CoV-2 variant pseudoviruses in female mice and shows comparable or equal potency against the wild-type strain, demonstrating that LSC-Exo may act as a broad-spectrum protectant against existing and emerging virus variants.

Editorial Note: Figure on page 11 in this Peer Review File has been amended to remove third-party material where no permission to publish could be obtained.

Reviewers' Comments:
Reviewer #1: Remarks to the Author: In this manuscript, Cheng and co-authors reported a comprehensive study to prevent broad SARS-CoV-2 infections using lung spheroid cell exosomes.The authors developed and synthesized the human lung spheroid cells (LSC)-derived exosomes (LSC-Exo) with high ACE2-expressing, enabling the LSC-Exo bind and neutralize SARS-CoV-2 and protect the host against the SARS-CoV-2 infection.They showed both in vitro and in vivo results that improved neutralizing capacity against pseudoviruses and elucidated the protective mechanisms.Overall, this is an exciting work with important material development and biological findings.Below are minor comments.
1.In Fig. 1d, TEM images of exosomes could be improved with higher resolution to visualize particle morphology and size.2. In Fig. 2, the authors showed the biodistribution of exosomes in the lungs.After nebulization, it would be helpful to assess the biodistribution of exosomes in other organs.What are the concentrations of exosomes and liposomes used in the analysis of in vivo distribution?Additionally, please add a scale bar in Fig. 2d. 3. The authors applied concentration equivalents in Fig. 3d,e to evaluate the effectiveness of neutralization at the cellular level.How many exosomes were employed here?4. The authors quantified relevant lung tissue markers, such as positive SARS-N cell and lung fibrosis, in Fig. 4i and j.It would be helpful to provide additional details about the procedures and tools used in Methods. 5. Some figures are in low resolution.6. Please include additional discussion regarding the future clinical translation of this platform.
Reviewer #2: Remarks to the Author: The manuscript reports on the use of exosomes derived from human lung spheroid cells to suppress SARS-CoV-2 infection in vitro and in vivo (mouse and hamster model).Exosomes from human LSCs contained more ACE2 than exosomes from HEK cells.DsRed uploaded in LSC exosomes reached the bronchioles more efficiently than dsRed uploaded in liposomes after inhalation of such exosomes or liposomes by mice using a Pari nebulizer.CD1 mice pre-exposed to inhaled LSC exosomes and 2h later infected with GFP expressing Baculovirus pseudotyped with SARS-CoV-2 D614G or luciferase and RFP expressing lentivirus pseudotyped with delta spike showed reduced reporter gene expression compared with mice that had been pretreated with nebulized PBS, recombinant ACE2, and HEK exosomes.Hamsters that were exposed to inhaled LSC exosomes and 2h later challenged with authentic SARS-CoV-2 presented with reduced body weight loss, viral replication and histopathology scores compared with hamsters that had been pre-exposed to PBS.Bulk RNA seq analysis of the lung tissues of the challenged hamsters, revealed a transcriptional pattern for the LSC exosome treated hamster that was closer to that of a sham-treated hamster than for the PBS treated and challenged hamsters.The authors have a very well established track record in the development of exosome for therapeutic use.Inhalation of ACE2 containing liposomes as a prophylaxis against SARS-CoV-2 infection is novel.Some experiments, however, require additional controls to support the conclusions.
Major remarks: 1. Exosomes were prepared by passing the 0.22 um filtrate of spent cell culture medium over a 100 kDa Amicon filter and detaching "the remaining exosomes" from the filter.The field seems to evolve to the implementation of rigorous separation and quality control methods to isolate extracellular vesicles (see e.g.PMID: 32854228).Mass spectrometry based QC of the prepared exosomes would be recommended.
2. Line 95: ACE2-deficient HEK293T cells: according to figure 1c, f and g, these cells do express ACE2.Please adapt the statement.3. It is not clear how the mice were exposed to nebulized materials.Was this done with a specially dimensioned snout-fitting nebulizer?It is stated that nebulization was with 10E9 particles per mouse.How was this dose determined? 4. In the comparison of the biodistribution, RFP-loaded HEK cell-derived exosomes should be included as controls.In addition, equal RFP uploading of exosomes and liposomes should be documented.5. Outbred CD1 mice were used.These mice are not susceptible to SARS-CoV-2 infection because the spike of human SARS-CoV-2 viruses does not recognize mouse ACE2, at least not the D614G or delta spike used here.How do the authors explain the reporter gene expression in control (PBS) treated mice in figure 3 h-k and in figure 6 e-h?In addition, the bar graphs in figure 3h and figure 6e don't appear to correspond with the presented whole lung images in those panels.Please explain.6.It is difficult to interpret figure 3a.What does the blue wavy line in the ACE2(?) decorated particles represent?Which symbol represents rhACE2?Are there large and small exosomes?Figure 3: the SARS-CoV-2 virion seems to contain a dsRNA genome whereas this is a positive-stranded RNA virus.Figure 3d and e: the X-axis represents concentrations.How was the concentration of the exosome preparations determined? 7. The hamster challenge experiment lacks a control treatment with HEK-derived exosomes.
Other remarks: 1.The publication by Ching et al., (ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection, 2022) should be mentioned in the discussion.2. Line 74: NCT04252167 does not seem to be registered at clinicaltrials.gov.3. Is the mouse monoclonal antibody that was raised against human MxA cross-reactive with hamster Mx1 (cfr.figure 5 b and c)? 4. Line 281: what is TNF-beta? 5. Figure 1d, legend: Please check the size of the scale bar.6. Line 394: please replace "mainly" by "only".7. Line 401: it is not clear that LSC-exo administration alone results in antioxidant activity, cfr."intrinsic".It appears that transcriptome analysis was performed on SARS-CoV-2 infected hamsters that had been pre-exposed to LSC-exo.8. Line 454, please specify the SARS-CoV-2 challenge strain that was used for the hamster experiment.
Reviewer #3: Remarks to the Author: "Inhalation of ACE2-expressing lung spheroid cell exosomes for prophylaxis of broad SARS-CoV-2 infection", by Dr Chang and collegues (manuscript number NCOMMS-22-49185) In this study, the Wang et al investigate the potential of ACE2-expressing human lung spheroid cells (LSC) derived extracellular vesicles to neutralize SARS-CoV-2 and inhibit the infection of multiple SARS-CoV-2 variants.For this, hACE2-expressing LSC-Exo were administered by nebulization.Biodistribution, retention and neutralization efficacy of LSC-Exo was studied in mice, while their prophylactic capacity against SARS-CoV-2 infection was assessed in Syrian hamster.They showed convincingly that ACE2-expressing LSC-Exo, but not ACE2-negative EVs, were able to bind and neutralize SARS-CoV-2 as well as SARS-CoV-2 variants of concern in vitro and in vivo (mice).The prophylactic activity of LSC-Exo against SARS-CoV-2 infection was demonstrated in Syrian hamster and an explanation for the underlying protection mechanisms provided.The manuscript addresses a timely topic.The presented data are sound and support the conclusions drawn.The authors critically discuss their findings and address the limitations of their study.In summary, this is a very well-conducted scientific study that shows ACE2-expressing extracellular vesicles as an interesting therapeutic approach for the treatment of SARS-CoV-2 infection.

Comments:
1) The biodistribution and neutralization efficacy was assessed in mice but not in Syrian hamsters?Are data available in hamsters as well?
2) Controls in the experiments with Syrian hamster: PBS, instead of ACE2-negative EVs, was used in the control group.Furthermore, a control group with LSC-Exo without subsequent SARS-CoV-2 infection is missing.Other EV cargo (proteins, nucleic acids) may may have an effect as well?Human LSC-EVs may affect the immune response/transcriptome in Syrian hamster even in the absence of SARS-CoV-2 infection.
Minor comments: 3) Based on the method of EV isolation, it is unlikely that solely exosomes, which are per definition extracellular vesicles of endosomal origin, were purified.The term small EVs (sEVs) would be more accurate, as recommended by the MISEV guidelines (https://doi.org/10.1080/20013078.2018.1535750).4) Fig. 1d: The TEM images are of poor quality and hardly allow an assessment of the morphology.Additional data on the characteristics and purity of the isolated EVs should be provided according to the MISEV guidelines.5) Fig. 3d,e: The dose is provided in ng/µL.LSC-and HEK-Exo samples contain other proteins as well and thus are not 'pure' ACE-2 proteins when compared to rhACE2.It is assumed that rhACE2 contains more ACE2 than LSC-Exo.Does the protein concentration correlate with the EV particle concentration?Are LSC-Exo and HEK-Exo comparable in this respect?6) Fig. 6k: Cytokine arrays from serum of mice treated with rhACE2 and HEK-Exo: HEK-Exo closer to sham or to the LSC-Exo group?
2. In Fig. 2, the authors showed the biodistribution of exosomes in the lungs.After nebulization, it would be helpful to assess the biodistribution of exosomes in other organs.What are the concentrations of exosomes and liposomes used in the analysis of in vivo distribution?Additionally, please add a scale bar in Fig. 2d.Re: We thank the reviewer for these great suggestions.The biodistribution of exosomes in other organs were studied.As shown in the new Fig.S5, in addition to lung, we observed that HEK-Exo and Liposome signals were starting to be shown in liver, spleen, and kidneys at 4 hours postinhalation (Fig. S5a, b).Comparatively, the LSC-Exo signals beginning to appear in major organs at 24 hours post-inhalation.These results could be attributed to the homologous targeting effects of LSC-Exo to lungs (as those exosomes were derived from lung cells).Confocal images of 24 hours post-inhalation exhibited that LSC-Exo, HEK-Exo and liposomes were detected in the heart, liver, spleen and kidneys (Fig. S5c), which might be attributed to the translocation of nanoparticles from the pulmonary tree to the bloodstream followed by delivery to other major organs through circulation.For the concentrations of exosomes and liposomes used in the analysis of in vivo distribution depended on the RFP loading within the exosomes and liposomes.In this manuscript, 10 9 LSC-Exo per mouse was used for the biodistribution analysis in mice.Per to the determination of RFP fluorescence at 595 nm, the RFP encapsulation efficiency of RFP-LSC was calculated to be 20.43%.The corresponding encapsulation efficiency of RFP-Lipo and RFP-HEK were determined to be 24.57% and 19.35%, respectively.As such, 0.83×10 9 RFP-Lipo and 1.05×10 9 RFP-HEK were used for per mouse.We have added this information in the revised manuscript.In addition, the scale bar in Fig. 2d has been added.3. The authors applied concentration equivalents in Fig. 3d,e to evaluate the effectiveness of neutralization at the cellular level.How many exosomes were employed here?Re: We thank the reviewer for this question.The neutralization assay was performed at 96-well plates with 100 µL cell medium, and the highest concentration of used in this assay is 100 ng/µL.Correspondingly, the numbers of LSC-Exo and HEK-Exo used for the highest concentration were 3.7e10 8 and 4.27e10 8 , respectively.4. The authors quantified relevant lung tissue markers, such as positive SARS-N cell and lung fibrosis, in Fig. 4i and j.It would be helpful to provide additional details about the procedures and tools used in Methods.Re: We thank the reviewer for this great suggestion.The procedures and tools for how to quantify the lung tissue markers were provided in the revised Methods.Here are what we stated: Histopathology and immunohistochemistry in infected hamsters Tissues were fixed with 4% PFA for 24 hours and transferred to 70% ethanol.The samples were paraffin embedded and the blocks were sectioned at a thickness of 5 µm.Slides were baked for 1 hour at 65 °C, deparaffinized in xylene, and rehydrated by a series of graded ethanol to distilled water.Subsequently, the slides were stained with hematoxylin (HSS16, Sigma-Aldrich) and eosin Y (318906, Sigma-Aldrich).Trichrome (HT10516, Sigma-Aldrich) assay was conducted according to the instructions of the manufacturer.Optical microscopy was performed to analyze these slides.Lung fibrosis was scored using the Ashcroft scale based on H&E staining, which uses a numerical scale from 0 through 8 to grade fibrosis according to previous report. 52

Some figures are in low resolution.
Re: In the SI, the high-resolution Figures were provided.In the manuscript, we will provide the .aiformat Figures to editor upon publication, ensuring they have high resolution.

Please include additional discussion regarding the future clinical translation of this platform.
Re: We thank the reviewer for this good suggestion.The additional discussion regarding the future clinical translation of our platform was provided in the revised manuscript.Here are what we stated: We envision that our ACE2-containing LSC-Exo could serve as a convenient and cost-effective agent to prevent initial infection or further internal dissemination of the virus, reduce viral transmission and alleviate disease onset of COVID-19.While this approach shows promise for clinical translation, several critical issues require careful consideration.It is essential to establish a harmonized approach to minimize batch-to-batch variation.Implementing rigorous quality control measures at each stage of the manufacturing process is necessary.Maintaining consistency in LSC-Exo production and ensuring homogeneity of their cargo are imperative goals.Moreover, additional steps, such as upscaling conditions, determining the appropriate culture medium for cell growth and expansion, evaluating the need for cell preconditioning, and developing conditioned medium production for LSC-Exo separation, must be addressed.

Reviewer #2
The manuscript reports on the use of exosomes derived from human lung spheroid cells to suppress SARS-CoV-2 infection in vitro and in vivo (mouse and hamster model).Exosomes from human LSCs contained more ACE2 than exosomes from HEK cells.DsRed uploaded in LSC exosomes reached the bronchioles more efficiently than dsRed uploaded in liposomes after inhalation of such exosomes or liposomes by mice using a Pari nebulizer.CD1 mice preexposed to inhaled LSC exosomes and 2h later infected with GFP expressing Baculovirus pseudotyped with SARS-CoV-2 D614G or luciferase and RFP expressing lentivirus pseudotyped with delta spike showed reduced reporter gene expression compared with mice that had been pretreated with nebulized PBS, recombinant ACE2, and HEK exosomes.Hamsters that were exposed to inhaled LSC exosomes and 2h later challenged with authentic SARS-CoV-2 presented with reduced body weight loss, viral replication and histopathology scores compared with hamsters that had been pre-exposed to PBS.Bulk RNA seq analysis of the lung tissues of the challenged hamsters, revealed a transcriptional pattern for the LSC exosome treated hamster that was closer to that of a sham-treated hamster than for the PBS treated and challenged hamsters.

The authors have a very well established track record in the development of exosome for therapeutic use. Inhalation of ACE2 containing liposomes as a prophylaxis against SARS-CoV-2 infection is novel. Some experiments, however, require additional controls to support the conclusions.
Re: We thank the reviewer for her/his comments that helped us substantially improve our manuscript.We have addressed each of the comments below and made revisions accordingly.Major remarks: 1. Exosomes were prepared by passing the 0.22 um filtrate of spent cell culture medium over a 100 kDa Amicon filter and detaching "the remaining exosomes" from the filter.The field seems to evolve to the implementation of rigorous separation and quality control methods to isolate extracellular vesicles (see e.g.PMID: 32854228).Mass spectrometry based QC of the prepared exosomes would be recommended.Re: We thank the reviewer for this great suggestion.According to the separation methods of extracellular vesicles in PMID: 32854228, the combination of ultrafiltration with TFF methods were utilized in our manuscript.The detailed experimental process was provided in the revised manuscript.
Here are what we stated: Exosomes were collected and isolated from LSC-Secretome via the combination of ultrafiltration with tangential flow filtration (TFF).Filtered secretomes were further filtered with 300 kDa, concentrated and washed with Dulbecco's phosphate-buffered saline (DPBS) through a KrosFlo ® KR2i TFF system (REPLIGEN, USA).The exosomes were filtered with a 0.22 µm filter to further remove cellular debris.After that, the collected exosomes were pipetted into a 100kDa Amicon centrifugal filter unit and centrifuged at 4000g at 4 °C.Once the medium was filtered, the remaining exosomes were collected from the filter and resuspended using DPBS with 25 mM Trehalose for further analysis.LSC-Exo and HEK-Exo were analyzed by nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical, Malvern, UK), western blot, Nanoimager (ONI, San Diego, USA) and Mass spectrometry.To analyze exosomal morphology, LSC-Exo and HEK-Exo were fixed onto copper grids and stained with vanadium negative staining for TEM (JEOL JEM-2000FX, Peabody, MA, USA).According to the reviewer's suggestion, we performed mass spectrometry on LSC-Exo and HEK-Exo.The new data is now available as Fig. S4.Venn diagram in Fig. S4a revealed that LSC-Exo and HEK-Exo share 2146 proteins.Further quantitative scatterplots analysis of these shared proteins (Fig. S4b) revealed that the top enriched proteins in LSC-Exo are A2MG, FNC, ALBU etc.We deduced that this enrichment was due to the use of fibronectin as a coating agent on the cell culture plate for LSC.We analyzed the exosomal biomarkers and identified the expression of CD9, CD63, CD81, TSG101, Alix, and VSP36 in both LSC-Exo and HEK-Exo (Fig. S4c).GO functional analysis indicated the proteins of HEK-Exo were mainly involved in RNA and DNA metabolic processes, whereas LSC-Exo were mainly involved in the extracellular matrix organization, response to growth factor and response to wounding etc (Fig. S4d).KEGG analysis revealed that mRNA surveillance pathway, Ras signaling pathway and cell cycle etc were enriched in HEK-Exo.In comparison, cytokine-cytokine receptor interaction, TGF-beta and NOD-like receptor signaling pathway etc. were enriched for LSC-Exo (Fig. S4e).These results suggest the successful isolation of LSC-Exo, which meets QC standards and exhibits a higher anti-inflammatory activity compared to HEK-Exo.Re: We thank the reviewer for this great suggestion.We have revised our statement as below: HEK293T cells with low ACE2 expression.3. It is not clear how the mice were exposed to nebulized materials.Was this done with a specially dimensioned snout-fitting nebulizer?It is stated that nebulization was with 10E 9 particles per mouse.How was this dose determined?
Re: We thank the reviewer for these great questions.Pari Trek S Portable 459 Compressor Nebulizer Aerosol System (047F45-LCS, PARI, Starnberg, Germany) was employed in our manuscript.This nebulizer is designed for multiple mice instead of being snout-fitted for individual mice, which was shown in Fig. 1 for reviewer only.
As the reviewer mentioned, the concentration of LSC-Exo nebulized for mice is 10E 9 particles per mouse, we dispersed 10E 9 of LSC-Exo particles into 1 mL PBS buffer and then this nebulizer was used to deliver LSC-Exo solution in the form of a fine into mouse.
[Redacted] Re: We thank the reviewer for pointing these out.We apologize for not providing enough experimental details.In mice, the CD1 mice were transduced with adenoviral vector expressing hACE2 (Ad5 -hACE2, VectorBuilder) firstly.Five days after Ad5-hACE2 transduction, mice were employed to perform the neutralization ability of LSC-Exo against SARS-CoV-2 pseudovirus and variants pseudovirus in mice.We have made it clear in the revised manuscript.As for the second question, we speculate that the mismatch between bar graphs in Fig. 3h and Fig. 6e with the presented whole lung images, may be attributed to the autofluorescence interference caused by residual blood in the lung tissues following abdominal aortic phlebotomy sacrifice.3a.What does the blue wavy line in the ACE2(?) decorated particles represent?Which symbol represents rhACE2?Are there large and small exosomes?Figure 3: the SARS-CoV-2 virion seems to contain a dsRNA genome whereas this is a positivestranded RNA virus.Figure 3d and e: the X-axis represents concentrations.How was the concentration of the exosome preparations determined?Re: In the Fig. 3a, the blue wavy line in the LSC-Exo or HEK-Exo represents the enclosed miRNA.The rhACE2 symbol was not shown in Fig. 3a.The large exosomes represent those exosomes close to RBD and the small exosomes represent those far from the RBD.In Fig. 3, we utilized SARS-CoV-2 pseudovirus to perform cell experiments.This SARS-CoV-2 pseudovirus is a HIV-based lentivirus, which indeed contains a dsRNA genome.In Fig. 3d, e, this neutralization assay was performed at 96-well plates with 100 µL cell medium, and the highest concentration of exosomes employed in this assay was 100 ng/µL.In our manuscript, the concentration of 10 10 exosome is about 270 µg which determined by microBCA protein assay kit, therefore, the 3.7e10 8 exosomes were used for the highest concentration.

The hamster challenge experiment lacks a control treatment with HEK-derived exosomes.
Re: We thank the reviewer for this great suggestion.The HEK-derived exosomes (HEK-Exo) control group has been added in the hamster challenge experiment.The results, as depicted in new Fig. 4 and Fig. 5, encompassing weight loss, viral load in both oral swabs and BAL, RNAscope assay, IHC staining of SARS-N, histological analysis and viral load assay of major organs, revealed that HEK-Exo treatment has little effect on decreasing viral load in oral swabs, BAL and major organs of hamsters as well as attenuating severe pneumonia caused by SARS-CoV-2.Re: We thank the reviewer for her/his comments that helped us substantially improve our manuscript.
We have addressed each of the comments below and made revisions accordingly.

Comments:
1.The biodistribution and neutralization efficacy was assessed in mice but not in Syrian hamsters?Are data available in hamsters as well?Re: We thank the reviewer for pointing these out.The biodistribution of LSC-Exo in Syrian hamsters after inhalation were studied.We found that LSC-Exo were predominantly localized in the lungs of hamsters 2 hours after inhalation (Fig S7).In contrast, after 24 hours of inhalation, LSC-Exo exhibited substantial distribution throughout the major organs of the hamsters.These results were consistent with the biodistribution results of LSC-Exo in mice.
To evaluate the neutralization capability of LSC-Exo against SARS-CoV-2 in hamsters, we infected the hamsters by SARS-CoV-2 firstly.After 24 hours, hamsters were inhaled with three doses of LSC-Exo on days 1, 2, and 3 post-challenge to investigate the potential of LSC-Exo in neutralizing SARS-CoV-2 WA1 infection (Fig. S16a).High levels of SARS-CoV-2 viral load were observed in the OS for both PBS and LSC-Exo treatment at on 2 nd day post-challenge (dpi), whereas a significant decrease in the viral load in OS was observed in the LSC-Exo group at 4, 7 days dpi compared to PBS group (Fig. S16b).Consistent with OS results, BAL viral load was approximately 4.136 log10 RNA copies per mL in the LSC-Exo group, which was lower than that in the PBS (5.23) group, indicating that LSC-Exo was capable of neutralizing SARS-CoV-2 (Fig. S16c).RNAscope analysis demonstrated that LSC-Exo efficiently repressed the viral replication (Fig. S16d).IHC analysis further demonstrated a pronounced inhibition of SARS-N expression in response to LSC-Exo treatment.(Fig. S16e).Histological analysis revealed that SARS-CoV-2-induced pulmonary hemorrhage and edema, as well as the significant infiltration of immune cells, were effectively mitigated by LSC-Exo treatment (Fig. S16f).Additionally, the levels of viral genomic RNA (Fig. S16g) and subgenomic RNA (Fig. S16h) in tissues such as the heart, liver, spleen, kidneys, and lymph nodes exhibited a significant reduction in hamsters treated with LSC-Exo.These data suggested that LSC-Exo has the capacity to neutralize SARS-CoV-2 as observed in mice and further block SARS-CoV-2 infection in hamster.Additionally, a control hamster group with LSC-Exo but without SARS-CoV-2 infection was added according to the reviewer's suggestion.The hamsters were inhaled with LSC-Exo and sacrificed 7 days after inhalation.Clinical chemistry and complete blood count (CBC) analyses were performed to assess whether human LSC-Exo could affect the immune response of Syrian hamster when SARS-CoV-2 infection was absent.As depicted in Fig. S18, the results indicated there was no significant difference between PBS group and LSC-Exo group in clinical chemistry analysis.Of specifical note, our findings revealed that CBC parameters of hamsters inhaled with LSC-Exo remained within normal ranges, but certain CBC parameters, such as white blood cell count, neutrophil and lymphocyte count, were significantly decreased in the LSC-Exo group compared to the PBS group (Fig. S19).These results suggest that LSC-Exo possesses anti-inflammatory properties capable of reducing the immune responses in hamsters.Consequently, it is plausible that other exosome cargo (proteins, nucleic acids) may have a little contribution to the protective ability of LSC-Exo against SARS-CoV-2 infection in hamsters by diminishing the inflammatory response caused by SARS-CoV-2.Re: We agreed with the reviewer's opinion that it is unlikely that solely exosomes were purified since currently available EV purification methods seldom allow for complete separation of exosomes and ectosomes.Some studies reported that the size of exosomes is about 30-100 nm, however, some literatures indicated exosomes are small membranous vesicles of 30-150 nm diameter.In our manuscript, we employed 0.22 µm filter to remove the cell debris and large extracellular vesicles.Hence, the reviewer is correct that the small EVs is more accurate than exosomes for our manuscript, given its physical size characterization.
Recently, multiple studies have been dedicated to discriminating the specific biomarkers between exosomes and ectosomes.Proteomics analysis demonstrated that VPS24, VPS32 and VPS36 were exclusively identified in exosomes.In contrast, VSP37D was only detected in ectosomes. 1We also performed the proteomics analysis of both LSC-Exo and HEK-Exo by mass spectrometry.We detected the VPS36 protein, rather than VSP37D protein, indicating that most of extracellular vesicles we purified are exosomes.Re: We thank the reviewer for these great suggestions.The high-resolution of TEM images were provided, as shown in Fig. 1d.Both LSC-Exo and HEK-Exo show a cup-shape morphology.It is recommended by MISEV that at least three positive and one negative protein markers of exosomes should be conducted for exosome characterization.Three tetraspanins biomarkers were studied by nanoimager.We found that distinct CD9, CD63 and CD81 biomarkers on single LSC-Exo and HEK-Exo (Fig. 1e).The expression of the cytosolic biomarkers Alix in exosomes have been demonstrated and acted as a reference protein for studying ACE2 expression on exosomes (Fig. 1g).In addition, the expression of cytosolic marker TSG101 in exosomes compared to negative marker calnexin were demonstrated (Fig. S3).
In addition, we have supplemented all the details regarding our exosome purification methods in the revised manuscript.
Here are what we stated:

Exosome isolation and characterization
Exosomes were collected and isolated from LSC-Secretome via the combination of ultrafiltration with tangential flow filtration (TFF) system.Filtered secretomes were further filtered with 300 kDa, concentrated and washed with Dulbecco's phosphate-buffered saline (DPBS) through a KrosFlo ® KR2i TFF system (REPLIGEN, USA).The exosomes were filtered with a 0.22 µm filter to further remove cellular debris.After that, the collected exosomes were pipetted into a 100kDa Amicon centrifugal filter unit and centrifuged at 4000g at 4 °C.Once the medium was filtered, the remaining exosomes were collected from the filter and resuspended using DPBS

Fig. S5 .
Fig. S5.Biodistribution of LSC-Exo in mice after inhalation.(a) Ex vivo imaging of major organs of mice after RFP-LSC, RFP-HEK or RFP-Lipo inhalation at the indicated time.(b) Quantification of the integrated density of RFP fluorescence in major organs; n=3 per group.(c) Confocal images showing the biodistribution of RFP-LSC, RFP-HEK or RFP-Lipo in heart, liver, spleen and kidney tissues and quantitative results from heart, liver, spleen and kidney tissues.Scale bar, 50 µm.Data are mean ± s.q.Statistical analysis was performed by one-way ANOVA with Bonferroni correction.

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Fig. S4.Proteomics analysis of LSC-Exo and HEK-Exo.(a) Venn diagram of proteins identified in LSC-Exo and HEK-Exo.(b) Quantitative scatterplots analysis of shared proteins in LSC-Exo and HEK-Exo.(c) Peptide numbers of specific biomarkers of exosomes.(d) GO function analysis of LSC-Exo and HEK-Exo.(e) KEGG pathway enrichment of LEC-Exo and HEK-Exo.

Fig. 4 .New Fig. 5 .
Fig. 4. Protective effect of LSC-Exo against authentic SARS-CoV-2 infection in Syrian hamsters.(a) Time courses of LSC-Exo inhalation, viral challenge, and measurements.(b) Changes in body weight of hamsters over 1-week post-challenge.n=5.(c) Viral RNA in oral swabs (OS) from hamsters treated with LSC-Exo, HEK-Exo or PBS.n=5.(d) Viral RNA in bronchoalveolar lavage (BAL) fluid from hamsters treated with LSC-Exo, HEK-Exo or PBS at 7 days post-challenge.n=5.(e) RNAscope images revealing regional distribution and viral RNA levels in hamster lungs.Immunohistochemistry analysis of SARS-N protein in lung tissues of hamsters.Scale bar, 50 μm.(f) Quantification analysis of positive SARS-N cell percentages in lungs of hamster.n=15.(g) H&E images of representative lung sections of hamsters.Scale bar, 500 μm.(h) Masson's trichrome staining of lung sections of hamsters.Scale bar, 500 μm.(i) Ashcroft scoring analysis of lung fibrosis from challenged hamsters that performed blindly.n=5.(j) Spider web plot displaying histopathological scoring of lung damage, normalized to sham control (green).Viral genomic RNA levels (k) and sgRNA levels (l) in tissues of

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Fig. S7.Biodistribution of LSC-Exo in hamsters after inhalation.(a) Ex vivo imaging of major organs of hamsters 2 hours and 24 hours after RFP-LSC inhalation.(b) Quantification of the integrated density of RFP fluorescence in major organs; n=3 per group.(c) Confocal images showing the biodistribution of LSC-Exo in heart, liver, spleen and kidney tissues of hamsters.Scale bar, 50 µm.Data are mean±s.d.Statistical analysis was performed by one-way ANOVA with Bonferroni correction.

Controls in the experiments with Syrian hamster: PBS, instead of ACE2-negative EVs, was used in the control group. Furthermore, a control group with LSC-Exo without subsequent SARS-CoV-2 infection is missing. Other EV cargo (proteins, nucleic acids) may have an effect as well? Human LSC-EVs may affect the immune response/transcriptome in Syrian hamster even in the absence of SARS-CoV-2 infection.
Statistical analysis was performed by two-way ANOVA with Tukey's multiple comparisons (b, g and h) or two-tailed, unpaired Student's t-test (c and e). 2. Re: According to the reviewer's suggestion, the HEK-Exo with low-ACE2 expression as a control group were added in the hamster experiment, as shown in new Fig.4.Based on the results obtained from the weight loss assay, viral load measurements in both oral swabs and BAL, RNAscope assay, IHC staining of SARS-N, histological analysis, and viral load assays conducted on major organs, as presented in the new Fig.4, it is evident that HEK-Exo treatment has minimal impact on reducing viral load in oral swabs, BAL, and major organs of hamsters.And it does not effectively attenuate the severe pneumonia caused by SARS-CoV-2.